The present invention relates to an atomic force microscope and methods of operating that microscope to provide both topographic and recognition imaging, and more particularly to an atomic force microscope for detecting interactions between a probe and a sensed agent on the surface of a substrate to provide simultaneous topographic and recognition images.
It has long been recognized that the atomic force microscope can be made to be sensitive to specific chemical interactions between a probe tip and a surface. For example, Lee et al., “Sensing discrete streptavidin-biotin interactions with atomic force microscopy,” Langmuir 10:354-357 (1994), demonstrated specific binding between biotin and streptavidin using chemically modified cantilever probes. Another example of a specific interaction between chemically reactive groups is given by Kienberger et al., “Static and dynamical properties of single poly (ethylene glycol) molecules investigated by force microscopy,” Single Molecules 1:123-128 (2000).
A method for attaching antibodies to a scanned probe has been described by Hinterdorfer et al., “Detection and localization of individual antibody-antigen recognition events by atomic force microscopy,” Proc. Natl. Acad. Sci. (USA) 93: 3477-3481 (1996); “Force spectroscopy of anti-body-antigen recognition measured by scanning force microscopy.” Biophys. J. 74:186 (1998); and “A mechanistic study of the dissociation of individual antibody-antigen pairs by atomic force microscopy,” Nanobiology 4:39-50 (1998). This method has been used to characterize interactions between several antibody-antigen pairs. The method has also been used to characterize interactions between adhesive proteins (Baumgartner, Hinterdorfer et al., “Cadherin interaction probed by atomic force microscopy.” Proc. Natl. Acad. Sci. USA 97 (2000)) and between ligands and transporter molecules embedded in native protein membranes.
Those skilled in the art will recognize that the technique is quite general and applicable to any set of materials that bind to one another—receptors with their corresponding proteins, drugs with their ligands, and antibodies with antigens. Thus, the chemical bonded to the probe may be termed the sensing agent, while the chemical recognized on a sample surface may be termed the sensed agent.
In the prior art methods described above, single molecule interaction forces are measured with chemically modified cantilever probe tips in molecular recognition force spectroscopy (MRFS) experiments using so-called force distance cycles: Highly selective ligands (preferentially one per tip) are covalently attached to the tip-end as shown in FIG. 1A. Referring to FIG. 1A, a cantilever probe tip 1 is modified with a specific reagent (such as ethanolamine or aminopropyltriethoxysilane) to place reactive groups (such as amines) on the surface of the probe 2. An amine reactive group 3, attached to one end of a flexible 8-nm long crosslinker 4, tethers the crosslinker (which may be polyethylene glycol (PEG) to the probe. A second reactive group (for sulfurs in this case, pyridine dithioproprionate (PDP.)) 5 reacts with the thiolated surface of the sensing agent, as carried out with the thiolating agent N-succinimidyl 3-(acetylthio) proprionate (SATP) 6, for example.
In this way, the sensing agent, in this case an antibody 7, is held on the end of the crosslinker 4. This arrangement has the advantage that the tethered sensing agent (the antibody 7) is free to move to the extent that the crosslinker 4 is flexible, thereby allowing the sensing agent to align with its target sensed agent (in this case the specific antigen for the antibody) on the surface being probed. Binding of the sensing agent 7 on the tip 1 with a specific sensed agent on the surface can be observed in the force-distance curve obtained as the tip is scanned towards the surface and retracted as shown in FIG. 1B.
The tip 1 is moved towards the surface of a sample, which leads to the formation of a single receptor-ligand bond between the tethered antibody and specific antigen on the sample surface. The force curve on approach shows no sign of this bond formation (“trace” 8). However, on retraction of the tip, a characteristic curve is observed (“retrace” 9) showing an increasing attractive force as the crosslinker 4 is stretched until the bond is broken when the retraced distance equals the almost fully extended length of the crosslinker at 10. The characteristic shape in the retrace reflects the viscoelastic properties of the crosslinker 4 by which the antibody 7 is tethered to the tip 1.
In the prior art described thus far, the surface must be probed by carrying out force-distance curves at every point of potential interest. In Elings et al, U.S. Pat. No. 5,519,212, the patentees state that the interaction between an antibody and an antigen can be detected by changes in the oscillation of a vibrated tip, although there is no description of how this may be accomplished. Raab et al provided the first practical demonstration of antibody-antigen recognition in a scanned image (Raab, Han et al., “Antibody recognition imaging by force microscopy,” Nature Biotech. 901-905 (1999)). In this work, a dynamic force microscope was operated in MACMODE (a trademark of Molecular Imaging Corp.), a mode in which the tip motion is controlled by an applied magnetic field. This mode of operation is described in greater detail in Lindsay, U.S. Pat. Nos. 5,515,719 and 5,513,518 and in Han, Lindsay et al., “A magnetically-driven oscillating probe microscope for operation in liquids,” Appl. Phys. Letts. 69:4111-4114 (1996).
Raab, Han et al. describe that the tip was driven into oscillation with an amplitude similar to the length of the crosslinker (4 in FIG. 1A) used to tether an antibody to the end of the tip. The antibody was antilysozyme and the antigen on the substrate was lysozyme. Referring to FIG. 2A, when a bare tip 21 is used to image the lysozyme 22, images such as those in FIG. 2C are obtained. When a modified tip 23 with antilysozyme 24 is attached (as shown in FIG. 2B), the images of the lysozyme are greatly broadened and increased in apparent height as shown in the image in FIG. 2E. The difference in the appearance of the images is illustrated by the line scans in FIG. 2D. The trace 25 over lysozyme taken with the bare tip 21 is narrower and lower than the trace 26 taken with the antibody tip 23, reflecting the attachment of antibody to antigen and subsequent stretching of the crosslinker as described by Raab, Han et al.
Receptor-ligand recognition is monitored by an enhanced reduction of the oscillation amplitude as a result of antibody-antigen binding. These binding signals are visible as bright and wide dots in the recognition image and reflect the position of ligand binding-sites with nanometer (nm) lateral accuracy. The drawback to this methodology is that the antibody-enzyme binding signals in the recognition image are interfered with by signals owing to the topographic features of the enzyme. Topography and recognition images can only be recorded by comparing a pair of images taken with bare and antibody-conjugated tips, respectively, and are, therefore, not obtained at the same time.
An increase in the speed of molecular recognition imaging is highly desirable, not just for increased effectiveness of microscopy, but also because a rapid molecular recognition method would enable very many small titer wells to be examined for binding affinity, opening a route for rapid drug screening. Accordingly, the need exists in the art to provide an atomic force microscope and method of operation that provides separate yet simultaneous topography and recognition images. A need also exists for a method for the rapid quantitative measurement of molecular binding with high spatial resolution.